The molten globule, a conformational ensemble with significant secondary structure but only loosely packed tertiary structure, has been suggested to be a ubiquitous intermediate in protein folding. However, it is difficult to assess the tertiary packing of transiently populated species to evaluate this hypothesis. Escherichia coli RNase H is known to populate an intermediate before the rate-limiting barrier to folding that has long been thought to be a molten globule. We investigated this hypothesis by making mimics of the intermediate that are the ground-state conformation at equilibrium, using two approaches: a truncation to generate a fragment mimic of the intermediate, and selective destabilization of the native state using point mutations. Spectroscopic characterization and the response of the mimics to further mutation are consistent with studies on the transient kinetic intermediate, indicating that they model the early intermediate. Both mimics fold cooperatively and exhibit NMR spectra indicative of a closely packed conformation, in contrast to the hypothesis of molten tertiary packing. This result is important for understanding the nature of the subsequent rate-limiting barrier to folding and has implications for the assumption that many other proteins populate molten globule folding intermediates.

The folding pathway of Escherichia coli RNase H is one of the best experimentally characterized for any protein. In spite of this, spectroscopic studies have never captured the earliest events. Using continuous-flow microfluidic mixing, we have now observed the first several milliseconds of folding by monitoring the tryptophan fluorescence lifetime (60 μs dead time). Two folding intermediates are observed, the second of which is the previously characterized I(core) millisecond intermediate. The new earlier intermediate is likely on-pathway and appears to have long-range non-native structure, providing a rare example of such non-native structure formation in a folding pathway. The tryptophan fluorescence lifetimes also suggest a deviation from native packing in the second intermediate, I(core). Similar results from a fragment of RNase H demonstrate that only half of the protein is significantly involved in this early structure formation. These studies give us a view of the formation of tertiary structure on the folding pathway, which complements previous hydrogen-exchange studies that monitored only secondary structure and observed sequential native structure formation. Our results provide detailed folding information on both a timescale and a size-scale accessible to all-atom molecular dynamics simulations of protein folding.

Tobacco smoke air pollution (TSAP) measurement may persuade parents to adopt smoke-free homes and thereby reduce harm to children from tobacco smoke in the home. In a pilot study involving 29 smoking families, a Sidepak was used to continuously monitor home PM(2.5) during an 8-h period, Sidepak and/or Dylos monitors provided real-time feedback, and passive nicotine monitors were used to measure home air nicotine for one week. Feedback was provided to participants in the context of motivational interviews. Home PM(2.5) levels recorded by continuous monitoring were not well-accepted by participants because of the noise level. Also, graphs from continuous monitoring showed unexplained peaks, often associated with sources unrelated to indoor smoking, such as cooking, construction, or outdoor sources. This hampered delivery of a persuasive message about the relationship between home smoking and TSAP. By contrast, immediate real-time PM(2.5) feedback (with Sidepak or Dylos monitor) was feasible and provided unambiguous information; the Dylos had the additional advantages of being more economical and quieter. Air nicotine sampling was complicated by the time-lag for feedback and questions regarding shelf-life. Improvement in the science of TSAP measurement in the home environment is needed to encourage and help maintain smoke-free homes and protect vulnerable children. Recent advances in the use of mobile devices for real-time feedback are promising and warrant further development, as do accurate methods for real-time air nicotine air monitoring.

Chromosome segregation begins when the cysteine protease, separase, cleaves the Scc1 subunit of cohesin at the metaphase-to-anaphase transition. Separase is inhibited prior to metaphase by the tightly bound securin protein, which contains a pseudosubstrate motif that blocks the separase active site. To investigate separase substrate specificity and regulation, here we develop a system for producing recombinant, securin-free human separase. Using this enzyme, we identify an LPE motif on the Scc1 substrate that is distinct from the cleavage site and is required for rapid and specific substrate cleavage. Securin also contains a conserved LPE motif, and we provide evidence that this sequence blocks separase engagement of the Scc1 LPE motif. Our results suggest that rapid cohesin cleavage by separase requires a substrate docking interaction outside the active site. This interaction is blocked by securin, providing a second mechanism by which securin inhibits cohesin cleavage.

Poverty threatens child health. In the United States, financial strain, which encompasses income and asset poverty, is common with many complex etiologies. Even relatively successful antipoverty programs and policies fall short of serving all families in need, endangering health. We describe a new approach to address this pervasive health problem: antipoverty medicine. Historically, medicine has viewed poverty as a social problem outside of its scope. Increasingly, health care has addressed povertys downstream effects, such as food and housing insecurity. However, strong evidence now shows that poverty affects biology, and thus, merits treatment as a medical problem. A new approach uses Medical-Financial Partnerships (MFPs), in which healthcare systems and financial service organizations collaborate to improve health by reducing family financial strain. MFPs help families grow assets by increasing savings, decreasing debt, and improving credit and economic opportunity while building a solid foundation for lifelong financial, physical, and mental health. We review evidence-based approaches to poverty alleviation, including conditional and unconditional cash transfers, savings vehicles, debt relief, credit repair, financial coaching, and employment assistance. We describe current national MFPs and highlight different applications of these evidence-based clinical financial interventions. Current MFP models reveal implementation opportunities and challenges, including time and space constraints, time-sensitive processes, lack of familiarity among patients and communities served, and sustainability in traditional medical settings. We conclude that pediatric health care practices can intervene upon poverty and should consider embracing antipoverty medicine as an essential part of the future of pediatric care.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant of concern evades antibody-mediated immunity that comes from vaccination or infection with earlier variants due to accumulation of numerous spike mutations. To understand the Omicron antigenic shift, we determined cryo-electron microscopy and x-ray crystal structures of the spike protein and the receptor-binding domain bound to the broadly neutralizing sarbecovirus monoclonal antibody (mAb) S309 (the parent mAb of sotrovimab) and to the human ACE2 receptor. We provide a blueprint for understanding the marked reduction of binding of other therapeutic mAbs that leads to dampened neutralizing activity. Remodeling of interactions between the Omicron receptor-binding domain and human ACE2 likely explains the enhanced affinity for the host receptor relative to the ancestral virus.

Analysis of the specificity and kinetics of neutralizing antibodies (nAbs) elicited by SARS-CoV-2 infection is crucial for understanding immune protection and identifying targets for vaccine design. In a cohort of 647 SARS-CoV-2-infected subjects, we found that both the magnitude of Ab responses to SARS-CoV-2 spike (S) and nucleoprotein and nAb titers correlate with clinical scores. The receptor-binding domain (RBD) is immunodominant and the target of 90% of the neutralizing activity present in SARS-CoV-2 immune sera. Whereas overall RBD-specific serum IgG titers waned with a half-life of 49 days, nAb titers and avidity increased over time for some individuals, consistent with affinity maturation. We structurally defined an RBD antigenic map and serologically quantified serum Abs specific for distinct RBD epitopes leading to the identification of two major receptor-binding motif antigenic sites. Our results explain the immunodominance of the receptor-binding motif and will guide the design of COVID-19 vaccines and therapeutics.

An ideal therapeutic anti-SARS-CoV-2 antibody would resist viral escape^1-3, have activity against diverse sarbecoviruses^4-7, and be highly protective through viral neutralization^8-11 and effector functions^12,13. Understanding how these properties relate to each other and vary across epitopes would aid the development of therapeutic antibodies and guide vaccine design. Here we comprehensively characterize escape, breadth and potency across a panel of SARS-CoV-2 antibodies targeting the receptor-binding domain (RBD). Despite a trade-off between in vitro neutralization potency and breadth of sarbecovirus binding, we identify neutralizing antibodies with exceptional sarbecovirus breadth and a corresponding resistance to SARS-CoV-2 escape. One of these antibodies, S2H97, binds with high affinity across all sarbecovirus clades to a cryptic epitope and prophylactically protects hamsters from viral challenge. Antibodies that target the angiotensin-converting enzyme 2 (ACE2) receptor-binding motif (RBM) typically have poor breadth and are readily escaped by mutations despite high neutralization potency. Nevertheless, we also characterize a potent RBM antibody (S2E12⁸) with breadth across sarbecoviruses related to SARS-CoV-2 and a high barrier to viral escape. These data highlight principles underlying variation in escape, breadth and potency among antibodies that target the RBD, and identify epitopes and features to prioritize for therapeutic development against the current and potential future pandemics.

SARS-CoV-2 can mutate and evade immunity, with consequences for efficacy of emerging vaccines and antibody therapeutics. Here, we demonstrate that the immunodominant SARS-CoV-2 spike (S) receptor binding motif (RBM) is a highly variable region of S and provide epidemiological, clinical, and molecular characterization of a prevalent, sentinel RBM mutation, N439K. We demonstrate N439K S protein has enhanced binding affinity to the hACE2 receptor, and N439K viruses have similar in vitro replication fitness and cause infections with similar clinical outcomes as compared to wild type. We show the N439K mutation confers resistance against several neutralizing monoclonal antibodies, including one authorized for emergency use by the US Food and Drug Administration (FDA), and reduces the activity of some polyclonal sera from persons recovered from infection. Immune evasion mutations that maintain virulence and fitness such as N439K can emerge within SARS-CoV-2 S, highlighting the need for ongoing molecular surveillance to guide development and usage of vaccines and therapeutics.